Cockayne Syndrome (CS) is an autosomal recessive disorder, characterized by growth failure, neurological abnormalities, premature aging symptoms, and cutaneous photosensitivity, but no increased cancer incidence. CS is divided into two strict complementation groups: CSA (mutation in CKN1) and CSB (mutation in ERCC6). Of the patients suffering from CS, 80% have mutations in the CSB gene. We are pursuing the hypothesis that the primary role of CS proteins is to facilitate the repair of endogenous DNA damage, and we have evidence for a direct role of CSB in regulating BER efficiency. Our in vitro work has also helped define the biochemical properties of CSB, revealing that the protein interacts with a diverse range of nucleic acid substrates and likely has important ATP-dependent and ATP-independent functions. More recent results, obtained in collaboration with Dr. Vilhelm Bohr, suggest that CSB plays a direct role in not only nuclear BER, but in mitochondrial BER, likely by helping recruit, stabilize, and/or retain BER proteins in repair complexes associated with the inner mitochondrial membrane. Moreover, CSB appears to act as a mitochondrial DNA damage sensor, inducing mitochondrial autophagy in response to stress, and thus, pharmacological modulators of autophagy are potential treatment options for this accelerated aging phenotype. CSB-deficient cells also exhibit a defect in efficient mitochondrial transcript production and the CSB protein specifically promotes elongation by the mitochondrial RNA polymerase suggesting that the pathologies associated with CS are in part, a direct result of the roles that CSB plays in mitochondria. Future work will aim to determine the biological substrates and molecular role(s) of the CS proteins in endogenous DNA damage repair. XRCC1 is a critical scaffold protein that orchestrates efficient single-strand break repair (SSBR), an important subpathway of BER. Recent data has found an association of XRCC1 with proteins causally linked to human spinocerebellar ataxias - aprataxin and tyrosyl-DNA phosphodiesterase 1 - implicating SSBR in protection against neuronal cell loss and neurodegenerative disease. In addition, molecular epidemiology studies in humans indicate that impaired function in XRCC1 may be associated with increased cancer susceptibility. We have evaluated a series of chronological and biological aging parameters in XRCC1 heterozygous (HZ) mice, which are deficient for XRCC1 function. HZ and wild-type (WT) C57BL/6 mice exhibit a similar median lifespan of 26 months and a nearly identical maximal life expectancy of 37 months. However, a number of HZ animals (7 of 92) showed a propensity for abdominal organ rupture, which may stem from developmental abnormalities given the prominent role of XRCC1 in endoderm and mesoderm formation. For other end-points evaluated weight, fat composition, blood chemistries, condition of major organs, tissues and relevant cell types, behavior, brain volume and function, and chromosome and telomere integrity HZ mice exhibited by-and-large a normal phenotype. Treatment of animals with the alkylating agent azoxymethane resulted in both liver toxicity and an increased incidence of precancerous lesions in the colon of HZ mice. Our study therefore indicates that XRCC1 haploinsufficiency in mammals has little effect on chronological longevity and many key biological markers of aging in the absence of environmental challenges, but may adversely affect normal animal development or increase disease susceptibility to a relevant genotoxic exposure. Ataxia with oculomotor apraxia 1 (AOA1) is caused by mutation in the APTX gene, which encodes the DNA stand break repair protein aprataxin. Aprataxin removes 5-adenylate groups in DNA that arise from aborted ligation reactions. AOA1 is characterized by global cerebellar atrophy, highlighted by loss of Purkinje cells, ocular motor apraxia, and motor and sensory neuropathy. Strikingly, AOA1 patients lack the cancer susceptibility and other peripheral symptoms (e.g., immunological deficiencies) commonly associated with other inherited disorders stemming from a DNA repair defect. We have found that aprataxin localizes to mitochondria in human cells, and have identified an N-terminal amino acid sequence that targets certain isoforms of the protein to this intracellular compartment. Interestingly, transcripts encoding this unique N-terminal stretch are expressed in the human brain, with highest production in the cerebellum. Depletion of aprataxin in human SH-SY5Y neuroblastoma cells and primary skeletal muscle myoblasts results in mitochondrial dysfunction, as revealed by reduced citrate synthase activity and mitochondrial DNA (mtDNA) copy number. Moreover, mtDNA, not nuclear DNA, has higher levels of background DNA damage upon aprataxin knockdown, suggesting a direct role for the enzyme in mtDNA processing. These data indicate that aprataxin activity is indispensable for maintaining mitochondrial function and that there likely is a mitochondrial component to the disease phenotype of AOA1. Future studies are aimed at determining the reason behind the tissue selectivity of the disorder. Expansion of CAG/CTG repeats in DNA is the underlying cause of >14 genetic disorders, including Huntington disease (HD) and myotonic dystrophy. The mutational process is ongoing, with increases in repeat size enhancing the toxicity of the expansion in specific tissues. In many repeat diseases, the repeats exhibit high instability in the striatum, whereas instability is minimal in the cerebellum. In recent work, we have provided molecular insights into how BER protein stoichiometry may contribute to the tissue-selective instability of CAG/CTG repeats by using specific repair assays. In particular, repair efficiency at CAG/CTG repeats and at control DNA sequences was markedly reduced under conditions that mimic the striatal situation, likely because of lower levels of the proteins APE1, FEN1, and LIG1. Moreover, damage located toward the 5' end of the repeat tract was poorly repaired, with the accumulation of incompletely processed intermediates as compared to an abasic lesion in the center or at the 3' end of the repeats or within control sequences. In addition, repair of lesions at the 5' end of CAG or CTG repeats involved multinucleotide synthesis, particularly at the cerebellar stoichiometry, suggesting that long-patch BER processes lesions at sequences susceptible to hairpin formation. Our results show that the BER stoichiometry, nucleotide sequence, and DNA damage position modulate repair outcome and suggest that a suboptimal long-patch BER activity promotes CAG/CTG repeat instability.